Compounds that Inhibit HIV Particle Formation

ABSTRACT

The present invention describes novel methods of identifying compounds which inhibit HIV particle formation and Rev-dependent HIV production. The present invention also provides methods and compounds for inhibiting HIV particle formation and or treating patients infected with HIV.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is entitled to priority pursuant to 35 U.S.C. §119(e)to U.S. provisional patent application Nos. 60/541,632, filed Feb. 4,2004, 60,569,354, filed May 7, 2004, 60/574,909, filed May 27, 2004, and60/583,173, filed Jun. 25, 2004.

US GOVERNMENT RIGHTS

This invention was made with United States Government support underGrant Nos. R21AI54213-01, and R21AI54213-02, awarded by NationalInstitutes of Health. The United States Government has certain rights inthe invention.

BACKGROUND

The Rev protein is absolutely essential for the production of the viralstructural proteins and thus inhibition of Rev function leads toinhibition of HIV replication through prevention of structural proteinsynthesis and the assembly of HIV particles (For a review on Rev seePollard, V. W., and M. H. Malim. 1998. Annu Rev Microbiol. 52:491-532).This is most easily shown using proviral clones that lack a functionalrev gene, in that they have no replicative ability (Feinberg et al 1986Cell 46:807-817; Bray et al. 1994 PNAS 91:1256-60). In the absence offunctional Rev, genomic HIV RNA and several other HIV mRNAs remain thecell nucleus (Felber et al. 1989 Proc Natl Acad Sci USA. 86:1496-1499;Hammarskjold et al. 1989, J. Virol. 63:1959-1966; Malim et al. Nature.338:254-257). Since the RNA is not exported to the cytoplasm, viralstructural proteins are not made and the infectious cycle cannotcontinue. Thus, it is clear that modalities inhibiting the function ofRev can form the basis for therapy against HIV infection and AIDS.

Although the Rev/RRE export pathway is still not fully understood,several important steps in this pathway have now been identified. Thecurrent understanding of this process is schematically shown in FIG. 1.The pathway starts with the import of Rev into the nucleus (step 1). Instep 2, Rev binds specifically to RNA containing a specific RNA elementcalled the Rev Response Element (RRE). In step 3, Rev multimerizes onthe RRE in a process believed to involve protein-protein as well asprotein-RNA interactions. In step 4, the Rev-RRE complex is recognizedby CRM1 and RAN-GTP, which initiates the export process and eventuallytargets the complex to the nuclear pore (Step 5), where it interactswith nucleoporins resulting in translocation to the cytoplasmic side ofthis complex (Step 6). Many details in the pathway have yet to beelucidated. Other soluble cellular proteins (e.g. EIF5A) may also play aspecific role in the Rev/RRE pathway. It is also not clear what happensonce the complex reaches the cytoplasm.

Viral replication inhibited by attacking required steps of Rev functiondescribed above has been demonstrated through the use of differentclasses of Rev mutants (Berger et al. 1991 Virology. 183:630-635).Mutants in each of the Rev functional domains have been described whichabolish viral replication. Additionally, a dominant negative form of theRev protein has been used to inhibit viral replication (Malim et al.1992. J Exp Med. 176:1197-1201).

The HIV virus particle consists of internal proteins that make up theviral core and two proteins that are part of the lipid envelope thatsurrounds the core. These proteins are expressed from precursormolecules called Pr55gag and Pr160gagpol for the core proteins and gp160for the envelope proteins. It is known that expression of these proteinsnormally requires co-expression of the HIV Rev protein. Without the Revprotein, the mRNAs encoding each of these proteins remains in thenucleus. In order for the Rev protein to work, it is also essential tohave an element present in the RNA that binds to Rev. This element iscalled the RRE.

There is a long felt need in the art for a better method to identifycompounds capable of inhibiting HIV replication and Rev function. Thepresent invention satisfies these needs.

SUMMARY OF THE INVENTION

The present invention is directed to novel methods of identifying HIVinhibitory compounds and the use of those compounds to treat patientsthat are HIV positive. The present invention is also directed to a safequantitative in vitro high-throughput assay to screen library compoundsfor effects on Rev-dependent p24 production. In one aspect, the methodsof the invention are directed to identifying inhibitors of HIVreplication.

There is clear evidence that Human Immunodeficiency Virus (HIV) is thecause of AIDS and that drugs that inhibit the replication and productionof infectious HIV particles are efficacious in the treatment of AIDS.This disclosure describes the identification of many compounds which arevery effective inhibitors of HIV particle formation. Without wishing tobe bound by any particular theory, the compounds may act by inhibitingHIV Rev function, HIV assembly, HIV particle budding or some other partof the HIV life cycle. The compounds are therefore likely to form thechemical basis for new drugs that could be used for the treatment ofAIDS.

The inhibitory compounds of the present invention were identified usingthe 5BD.1 cell line to screen for drugs that inhibit HIV particleformation without showing toxicity in a 5 day cell survival assay. Theamount of HIV particles released by budding from the 5BD.1 cell lineinto the culture medium was measured using a simple and straightforwardELISA assay. 40,000 compounds were screened and 12 were selected as“hits” based on their ability to inhibit HIV particle formation withoutshowing toxicity in a 5 day cell survival assay.

Various aspects and embodiments of the invention are described infurther detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates known steps in the Rev/RRE pathway.

FIG. 2 graphically summarizes the ELISA data readout from part of theprimary screen of 40,000 compounds plotted as a percentage of thecontrol. Compounds that gave an inhibition of HIV particle formationbelow 50% were chosen for further study. In the total screen, 192compounds gave values below 50%.

FIG. 3 is a graphic illustration of a dose response curve testing thecompounds 75168 (upper panel) and 89246 (lower panel) for effects on HIVparticle release from cell lines and on toxicity. The data illustrate asix concentration dose response experiment for the compounds added tothe cell line and are indicate measurement of p24 (diamonds) in 5BD.1cells and toxicity in MT4 cells (squares). Each compound is named withan identifier along the X-axis of the graph, as are the concentrationsof the compound tested. The left Y-axis indicates HIV particle releaseinto the medium as a percentage of the control (untreated). The rightY-axis indicates toxicity as a percentage of control (untreated) and avalue of 100% indicates no toxicity. into the medium as a percentage ofthe control (untreated). Details of the assay are given in part E.

FIG. 4, comprising FIGS. 4A to 4H, provides the chemical structures andcode name, Specs name, and chemical name of eight of the compoundsidentified in the assays of the invention, namely: 89246 (4A), 91161(4B), 103833 (4C), 104366 (4D), 107129 (4E), 107740 (4F), 75168 (4G),and 109020 (4H).

FIG. 5, comprising FIGS. 5A to 5C, is a graphic illustration of theresults of testing leptomycin B (control; upper panel; 5A), compound99246 (middle panel; 5B) and compound 104355 (lower panel; 5C) forinhibition of HIV-1 Rev (left ordinate) and for toxicity (rightordinate) using 5BD.1 cells. The concentration (μM) is indicated on theabscissa. Similar data were obtained for all the compounds depicted inFIG. 4 and were used to calculate IC₅₀ and TC₅₀.

FIG. 6, comprising FIGS. 6A to 6C, is a graphic illustration of a doseresponse curve indicating the effects of AZT (upper panel; 6A), compound104366 (middle panel; 6B), and compound 89246 (lower panel; 6C) in aPBMC viral replication assay with the primary isolate HIV-1 BR 93 021(closed diamonds). Toxicity data is also plotted (open squares). Similardata for all eight compounds was used to calculate the IC₅₀ and TC₅₀.

FIG. 7, comprising FIGS. 7A to 7C, graphically illustrates the responseto the AZT control (upper panel; 7A), compound 104366 (middle panel;7B), and compound 89246 (lower panel; 7C) with the lab isolate HIV-1NL4-3 (diamonds) in a PBMC viral replication assay. Toxicity data isalso plotted (squares). Similar data for all eight compounds was used tocalculate the IC₅₀ and TC₅₀. VC=viral control (diamonds); CC=cellcontrol (squares)

FIG. 8, comprising FIGS. 8A to 8C, graphically illustrates the doseresponse to the Temacrazine (TMZ) control (upper panel; 8A), compound89246 (middle panel; 8B), and compound 103833 (lower panel; 8C) in a U1latency activation assay (diamonds) and in a toxicity assay (squares).Concentration (μM) is indicated on the abscissa. Similar data for sevencompounds was obtained and used to calculate the IC₅₀ and TC₅₀.Ordinates represent percent of virus control (VC; left ordinate) andpercent of cell control (CC; right ordinate).

FIG. 9 schematically illustrates the structures of compound 89246 andfour of its analogs.

FIG. 10 schematically illustrates the structures of compound 91161 andthirteen of its analogs.

FIG. 11 schematically illustrates the structures of compound 103833 and29 of its analogs.

FIG. 12 schematically illustrates the structures of compound 104366 andsix of its analogs.

FIG. 13 schematically illustrates the structures of compound 107129 andsix of its analogs.

FIG. 14 schematically illustrates the structures of compound 107440 andsix of its analogs.

FIG. 15 schematically illustrates the structures of compound 109020 andeighteen of its analogs.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

As used herein, the articles “a” and “an” refer to one or to more thanone, i.e., to at least one, of the grammatical object of the article. Byway of example, “an element” means one element or more than one element.

As used herein, an “analog” of a chemical compound is a compound that,by way of example, resembles another in structure but is not necessarilyan isomer (e.g., 5-fluorouracil is an analog of thymine).

A disease or disorder is “alleviated” if the severity of a symptom ofthe disease or disorder, the frequency with which such a symptom isexperienced by a patient, or both, are reduced.

As used herein, a “derivative” of a compound refers to a chemicalcompound that may be produced from another compound of similar structurein one or more steps, as in replacement of H by an alkyl, acyl, or aminogroup.

As used herein, a “functional” biological molecule is a biologicalmolecule in a form in which it exhibits a property or activity by whichit is characterized. A functional enzyme, for example, is one whichexhibits the characteristic catalytic activity by which the enzyme ischaracterized.

“Inhibiting HIV replication” as used herein, refers to any method ortechnique which inhibits HIV particle formation, virion production,release, as well as methods the induction of such processes. The termincludes inhibiting Rev expression, function, or activity.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the composition of the inventionfor its designated use. The instructional material of the kit of theinvention may, for example, be affixed to a container which contains thecomposition or be shipped together with a container which contains thecomposition. Alternatively, the instructional material may be shippedseparately from the container with the intention that the instructionalmaterial and the composition be used cooperatively by the recipient.

As used herein, a “ligand” is a compound that specifically binds to atarget compound or molecule. A ligand “specifically binds to” or “isspecifically reactive with” a compound when the ligand functions in abinding reaction which is determinative of the presence of the compoundin a sample of heterogeneous compounds.

As used herein, the term “purified” and like terms relate to anenrichment of a molecule or compound relative to other componentsnormally associated with the molecule or compound in a nativeenvironment. The term “purified” does not necessarily indicate thatcomplete purity of the particular molecule has been achieved during theprocess. A “highly purified” compound as used herein refers to acompound that is greater than 90% pure.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil emulsion, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans.

A “subject” of diagnosis or treatment is a mammal, including a human.

As used herein, the term “treating” includes prophylaxis of the specificdisorder or condition, or alleviation of the symptoms associated with aspecific disorder or condition and/or preventing or eliminating saidsymptoms. A “prophylactic” treatment is a treatment administered to asubject who does not exhibit signs of a disease or exhibits only earlysigns of the disease for the purpose of decreasing the risk ofdeveloping pathology associated with the disease.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology for the purpose of diminishing oreliminating those signs.

A “therapeutically effective amount” of a compound is that amount ofcompound which is sufficient to provide a beneficial effect to thesubject to which the compound is administered.

CHEMICAL DEFINITIONS

As used herein, the term “halogen” or “halo” includes bromo, chloro,fluoro, and iodo.

The term “haloalkyl” as used herein refers to an alkyl radical bearingat least one halogen substituent, for example, chloromethyl, fluoroethylor trifluoromethyl and the like.

The term “C₁-C_(n) alkyl” wherein n is an integer, as used herein,represents a branched or linear alkyl group having from one to thespecified number of carbon atoms. Typically C₁-C₆ alkyl groups include,but are not limited to, methyl, ethyl, n-propyl, iso-propyl, butyl,iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl and the like.

The term “C₂-C_(n) alkenyl” wherein n is an integer, as used herein,represents an olefinically unsaturated branched or linear group havingfrom 2 to the specified number of carbon atoms and at least one doublebond. Examples of such groups include, but are not limited to,1-propenyl, 2-propenyl, 1,3-butadienyl, 1-butenyl, hexenyl, pentenyl,and the like.

The term “C₂-C_(n) alkynyl” wherein n is an integer refers to anunsaturated branched or linear group having from 2 to the specifiednumber of carbon atoms and at least one triple bond. Examples of suchgroups include, but are not limited to, 1-propynyl, 2-propynyl,1-butynyl, 2-butynyl, 1-pentynyl, and the like.

The term “C₃-C_(n) cycloalkyl” wherein n=8, represents cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

As used herein the term “aryl” refers to a mono- or bicyclic carbocyclicring system having one or two aromatic rings including, but not limitedto, phenyl, benzyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, andthe like.

The term (C₅-C₈ alkyl)aryl refers to any aryl group which is attached tothe parent moiety via the alkyl group.

The term “heterocyclic group” refers to a mono- or bicyclic carbocyclicring system containing from one to three heteroatoms wherein theheteroatoms are selected from the group consisting of oxygen, sulfur,and nitrogen.

As used herein the term “heteroaryl” refers to a mono- or bicycliccarbocyclic ring system having one or two aromatic rings containing fromone to three heteroatoms and includes, but is not limited to, furyl,thienyl, pyridyl and the like.

The term “bicyclic” represents either an unsaturated or saturated stable7- to 12-membered bridged or fused bicyclic carbon ring. The bicyclicring may be attached at any carbon atom which affords a stablestructure. The term includes, but is not limited to, naphthyl,dicyclohexyl, dicyclohexenyl, and the like.

The term “pharmaceutically-acceptable salt” refers to salts which retainthe biological effectiveness and properties of the S1P analogs of thepresent invention and which are not biologically or otherwiseundesirable. In many cases, the S1P analogs of the present invention arecapable of forming acid and/or base salts by virtue of the presence ofamino and/or carboxyl groups or groups similar thereto.

EMBODIMENTS

In one embodiment, the present invention is directed to a method ofidentifying compounds, and their analogs and derivatives, which inhibitHIV replication. HIV replication and HIV particle formation are usedsynonymously herein. In one aspect, the invention is directed to amethod of identifying compounds with anti-rev activity. In one aspect,the invention is useful for screening many known compounds. In anotheraspect, the invention is useful for identifying activity of unknowncompounds in mixtures such as cell or plant extracts.

In one aspect, the invention describes compounds, and analogs andderivatives thereof, which inhibit HIV particle formation by inhibitingRev function or activity. One of ordinary skill in the art wouldunderstand that there are many methods to measure Rev function oractivity. Methods and techniques for measuring Rev function and activityare useful for determining whether a compound inhibits HIV replication.Those methods not described herein are known in the art and areavailable to those of ordinary skill in the art. The assays describedherein are useful for identifying inhibitors of HIV replication, whetherthe assay measures replication directly or indirectly.

In one aspect of the invention, a cell which produces HIV virions in aRev-protein dependent fashion is contacted with a test compound.Inhibition of HIV replication in said cells, relative to HIV replicationin cells treated with the test compound which produce HIV virions in aRev-independent fashion, is a an indication that said compound inhibitsRev-dependent HIV virion production. In one aspect of the invention,cell lines useful for identifying inhibitors of HIV replication include,but are not limited to, 5BD.1, 5BA.1, and 2A.22.

In accordance with one embodiment an HIV inhibitor is provided whereinthe compound has the general structure:

wherein

A is selected from the group consisting of N, CR₁, and

B is selected from the group consisting of N and S;

Y is selected from the group consisting of Se, CH and CR₄;

X is selected from the group consisting of CH and N;

R₁ is selected from the group consisting of H, NR₅R₆ and

R₂ and R₃ are independently selected from the group consisting of H,halo, hydroxy and C₁-C₄ alkyl;

R₄ is selected from the group consisting of H, halo, hydroxy and C₁-C₄alkyl;

R₅ and R₆ are independently selected from the group consisting of H andC₁-C₄ alkyl;

R₇ and R₈ are independently selected from the group consisting of H,halo, hydroxy, C₁-C₄ alkyl, C₁-C₄ alkoxy, —NHC(O)CH₃ and —O(C₁-C₄alkyl)(C₅-C₆ heterocyclic) or R₇ and R₈ together with the atoms to whichthey are attached form an optionally substituted C₅-C₆ aryl, wherein thearyl ring is optionally substituted with halo, C₁-C₄ alkyl, C₁-C₄alkoxy, C₁-C₄ alkyl(C₅-C₆ aryl) and —O(C₁-C₄ alkyl)(C₅-C₆ heterocyclic).In one embodiment Y is CR₄, R₇ is H or C₁-C₄ alkoxy, R₈ is halo or

wherein n is an integer ranging from 1-5, and P, W and Z areindependently selected from the group consisting of O, S, CH₂ and NH.

In another embodiment a compound is provided wherein the compound hasthe general structure:

wherein

X is selected from the group consisting of CH and N;

R₁ is selected from the group consisting of H, NR₅R₆ and

R₂ and R₃ are independently selected from the group consisting of H,halo, hydroxy and C₁-C₄ alkyl;

R₄ is selected from the group consisting of H, halo, hydroxy and C₁-C₄alkyl,

R₅ and R₆ are independently selected from the group consisting of H andC₁-C₄ alkyl;

R₇ and R₈ are independently selected from the group consisting of H,halo, hydroxy, C₁-C₄ alkyl, C₁-C₄ alkoxy, —NHC(O)CH₃ and —O(C₁-C₄alkyl)(C₅-C₆ heterocyclic) or R₇ and R₈ together with the atoms to whichthey are attached form an optionally substituted C₅-C₆ aryl, wherein thearyl ring is optionally substituted with halo, C₁-C₄ alkyl, C₁-C₄alkoxy, C₁-C₄ alkyl(C₅-C₆ aryl) and —O(C₁-C₄ alkyl)(C₅-C₆ heterocyclic).In one embodiment R₁ is NR₅R₆ or

R₇ is H or C₁-C₄ alkoxy, and

R₈ is halo or

wherein n is an integer ranging from 1-5, and P, W and Z areindependently selected from the group consisting of O, S, CH₂ and NH.

Another embodiment of the invention is directed to compounds of theinvention identified as inhibitors of Rev-activity, such as thefollowing eight compounds, as well as analogs, modifications, andderivatives thereof:

Yet another embodiment of the invention is directed to the compounds ofFIGS. 9, 10, 11, 12, 13, 14, and 15, which are analogs of the compoundslisted above (see also FIG. 4).

The present invention is also directed to pharmaceutical compositionscomprising the HIV inhibitory compounds of the present invention. Moreparticularly, such compounds can be formulated as pharmaceuticalcompositions using standard pharmaceutically acceptable carriers,fillers, solublizing agents and stabilizers known to those skilled inthe art.

The invention is also directed to methods of administering the compoundsof the invention to a subject. In one embodiment, the invention providesa method of treating a subject with HIV by administering compoundsidentified using the methods of the invention description. It ispreferred that a compound inhibits HIV replication by at least 10%relative to a control where a compound is not being used to inhibit HIVreplication. It is more preferred that a compound of the inventioninhibits HIV replication by at least 25% relative to untreated controls.It is further preferred that a compound of the invention inhibits HIVreplication by at least 50% relative to untreated controls. It is evenfurther preferred that a compound of the invention inhibits HIVreplication by at least 75% relative to untreated controls. It is alsopreferred that a compound of the invention inhibits HIV replication byat least 90% relative to untreated controls. In yet another aspect, itis preferred that a compound of the invention inhibits HIV replicationby at least 95% relative to untreated controls. In one aspect of theinvention, HIV replication is inhibited due to inhibition of Revfunction or activity. Pharmaceutical compositions comprising the presentcompounds are administered to an individual in need thereof by anynumber of routes including, but not limited to, topical, oral,intravenous, intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, intraperitoneal,intranasal, enteral, topical, sublingual, or rectal means.

In accordance with one embodiment, a method of treating HIV in a subjectin need such treatment is provided. The method comprises administering apharmaceutical composition comprising at least one HIV inhibitorycompound of the present invention to a patient in need thereof.Compounds identified by the methods of the invention which inhibit HIVreplication can be administered with known anti-HIV compounds or othermedications as well.

The invention also encompasses the use pharmaceutical compositions of anappropriate compound, analog, or derivative thereof to practice themethods of the invention, the composition comprising at least oneappropriate compound, analog, or derivative thereof and apharmaceutically-acceptable carrier.

As used herein, the term “pharmaceutically-acceptable carrier” means achemical composition with which an appropriate compound may be combinedand which, following the combination, can be used to administer theappropriate compound to a mammal. Preferably the mammal is a human.

The pharmaceutical compositions useful for practicing the invention maybe administered to deliver a dose of between 1 ng/kg/day and 100mg/kg/day.

Pharmaceutical compositions that are useful in the methods of theinvention may be administered systemically in oral solid formulations,ophthalmic, suppository, aerosol, topical or other similar formulations.In addition to the appropriate compound, such pharmaceuticalcompositions may contain pharmaceutically-acceptable carriers and otheringredients known to enhance and facilitate drug administration. Otherpossible formulations, such as nanoparticles, liposomes, resealederythrocytes, and immunologically based systems may also be used toadminister an appropriate compound according to the methods of theinvention.

Compounds which are identified using any of the methods described hereinmay be formulated and administered to a mammal for treatment of thediseases disclosed herein are now described.

The invention encompasses the preparation and use of pharmaceuticalcompositions comprising a compound useful for treatment of the diseasesdisclosed herein as an active ingredient. Such a pharmaceuticalcomposition may consist of the active ingredient alone, in a formsuitable for administration to a subject, or the pharmaceuticalcomposition may comprise the active ingredient and one or morepharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. The active ingredient may bepresent in the pharmaceutical composition in the form of aphysiologically acceptable ester or salt, such as in combination with aphysiologically acceptable cation or anion, as is well known in the art.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition with which the active ingredient may be combinedand which, following the combination, can be used to administer theactive ingredient to a subject.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of the active ingredient which is compatiblewith any other ingredients of the pharmaceutical composition, which isnot deleterious to the subject to which the composition is to beadministered.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, and dogs,birds including commercially relevant birds such as chickens, ducks,geese, and turkeys.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal,buccal, ophthalmic, intrathecal or another route of administration.Other contemplated formulations include projected nanoparticles,liposomal preparations, resealed erythrocytes containing the activeingredient, and immunologically-based formulations.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

In addition to the active ingredient, a pharmaceutical composition ofthe invention may further comprise one or more additionalpharmaceutically active agents. Particularly contemplated additionalagents include anti-emetics and scavengers such as cyanide and cyanatescavengers.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitablefor oral administration may be prepared, packaged, or sold in the formof a discrete solid dose unit including, but not limited to, a tablet, ahard or soft capsule, a cachet, a troche, or a lozenge, each containinga predetermined amount of the active ingredient. Other formulationssuitable for oral administration include, but are not limited to, apowdered or granular formulation, an aqueous or oily suspension, anaqueous or oily solution, or an emulsion.

As used herein, an “oily” liquid is one which comprises acarbon-containing liquid molecule and which exhibits a less polarcharacter than water.

A tablet comprising the active ingredient may, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets may be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycollate. Known surface active agentsinclude, but are not limited to, sodium lauryl sulphate. Known diluentsinclude, but are not limited to, calcium carbonate, sodium carbonate,lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to formosmotically-controlled release tablets. Tablets may further comprise asweetening agent, a flavoring agent, a coloring agent, a preservative,or some combination of these in order to provide pharmaceuticallyelegant and palatable preparation.

Hard capsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made usinga physiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which may be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition of the inventionwhich are suitable for oral administration may be prepared, packaged,and sold either in liquid form or in the form of a dry product intendedfor reconstitution with water or another suitable vehicle prior to use.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose.

Known dispersing or wetting agents include, but are not limited to,naturally-occurring phosphatides such as lecithin, condensation productsof an alkylene oxide with a fatty acid, with a long chain aliphaticalcohol, with a partial ester derived from a fatty acid and a hexitol,or with a partial ester derived from a fatty acid and a hexitolanhydride (e.g. polyoxyethylene stearate, heptadecaethyleneoxycetanol,polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitanmonooleate, respectively). Known emulsifying agents include, but are notlimited to, lecithin and acacia. Known preservatives include, but arenot limited to, methyl, ethyl, or n-propyl-para-hydroxybenzoates,ascorbic acid, and sorbic acid. Known sweetening agents include, forexample, glycerol, propylene glycol, sorbitol, sucrose, and saccharin.Known thickening agents for oily suspensions include, for example,beeswax, hard paraffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for rectal administration. Such acomposition may be in the form of, for example, a suppository, aretention enema preparation, and a solution for rectal or colonicirrigation.

Suppository formulations may be made by combining the active ingredientwith a non-irritating pharmaceutically acceptable excipient which issolid at ordinary room temperature (i.e. about 20° C.) and which isliquid at the rectal temperature of the subject (i.e. about 37° C. in ahealthy human). Suitable pharmaceutically acceptable excipients include,but are not limited to, cocoa butter, polyethylene glycols, and variousglycerides. Suppository formulations may further comprise variousadditional ingredients including, but not limited to, antioxidants andpreservatives.

Retention enema preparations or solutions for rectal or colonicirrigation may be made by combining the active ingredient with apharmaceutically acceptable liquid carrier. As is well known in the art,enema preparations may be administered using, and may be packagedwithin, a delivery device adapted to the rectal anatomy of the subject.Enema preparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants and preservatives.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for vaginal administration. Such acomposition may be in the form of, for example, a suppository, animpregnated or coated vaginally-insertable material such as a tampon, adouche preparation, or gel or cream or a solution for vaginalirrigation.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e. such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

Douche preparations or solutions for vaginal irrigation may be made bycombining the active ingredient with a pharmaceutically acceptableliquid carrier. As is well known in the art, douche preparations may beadministered using, and may be packaged within, a delivery deviceadapted to the vaginal anatomy of the subject. Douche preparations mayfurther comprise various additional ingredients including, but notlimited to, antioxidants, antibiotics, antifungal agents, andpreservatives.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e. powder or granular) form for reconstitution with asuitable vehicle (e.g. sterile pyrogen-free water) prior to parenteraladministration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer systems. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers, and preferably from about 1 toabout 6 nanometers. Such compositions are conveniently in the form ofdry powders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder or using a self-propelling solvent/powder-dispensingcontainer such as a device comprising the active ingredient dissolved orsuspended in a low-boiling propellant in a sealed container. Preferably,such powders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers. Morepreferably, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositionspreferably include a solid fine powder diluent such as sugar and areconveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic or solid anionic surfactant or a solid diluent(preferably having a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may also provide the active ingredient in the form of dropletsof a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration preferably have an averagediameter in the range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient and having an average particlefrom about 0.2 to 500 micrometers. Such a formulation is administered inthe manner in which snuff is taken i.e. by rapid inhalation through thenasal passage from a container of the powder held close to the nares.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and may further comprise one or more of theadditional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, 0.1 to 20% (w/w)active ingredient, the balance comprising an orally dissolvable ordegradable composition and, optionally, one or more of the additionalingredients described herein. Alternately, formulations suitable forbuccal administration may comprise a powder or an aerosolized oratomized solution or suspension comprising the active ingredient. Suchpowdered, aerosolized, or aerosolized formulations, when dispersed,preferably have an average particle or droplet size in the range fromabout 0.1 to about 200 nanometers, and may further comprise one or moreof the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for ophthalmic administration. Suchformulations may, for example, be in the form of eye drops including,for example, a 0.1-1.0% (w/w) solution or suspension of the activeingredient in an aqueous or oily liquid carrier. Such drops may furthercomprise buffering agents, salts, or one or more other of the additionalingredients described herein. Other opthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form or in a liposomal preparation.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed., 1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which isincorporated herein by reference.

Typically, dosages of the compound of the invention which may beadministered to an animal, preferably a human, range in amount from 1 μgto about 100 g per kilogram of body weight of the animal. While theprecise dosage administered will vary depending upon any number offactors, including but not limited to, the type of animal and type ofdisease state being treated, the age of the animal and the route ofadministration. Preferably, the dosage of the compound will vary fromabout 1 mg to about 10 g per kilogram of body weight of the animal. Morepreferably, the dosage will vary from about 10 mg to about 1 g perkilogram of body weight of the animal.

The compound may be administered to an animal as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even leesfrequently, such as once every several months or even once a year orless. The frequency of the dose will be readily apparent to the skilledartisan and will depend upon any number of factors, such as, but notlimited to, the type and severity of the disease being treated, the typeand age of the animal, etc.

The invention also includes a kit comprising the composition of theinvention and an instructional material which describes adventitiallyadministering the composition to a cell or a tissue of a mammal. Inanother embodiment, this kit comprises a (preferably sterile) solventsuitable for dissolving or suspending the composition of the inventionprior to administering the compound to the mammal.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the peptide of the invention inthe kit for effecting alleviation of the various diseases or disordersrecited herein. Optionally, or alternately, the instructional materialmay describe one or more methods of alleviation the diseases ordisorders in a cell or a tissue of a mammal. The instructional materialof the kit of the invention may, for example, be affixed to a containerwhich contains the peptide of the invention or be shipped together witha container which contains the peptide. Alternatively, the instructionalmaterial may be shipped separately from the container with the intentionthat the instructional material and the compound be used cooperativelyby the recipient.

EXAMPLES

The invention is now described with reference to the following examples.These examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseexamples, but rather should be construed to encompass any and allvariations which become evident as a result of the teachings providedherein.

There is clear evidence that Human Immunodeficiency Virus (HIV) is thecause of AIDS and that drugs that inhibit the replication and productionof infectious HIV particles are efficacious in the treatment of AIDS(Greene W. C. 2004 Nat. Immunol. 9:867-871). The compounds described inthis application are very effective inhibitors of HIV particle formationand are disclosed herein to inhibit HIV Rev function. Without wishing tobe bound by any particular theory, these compounds are likely to work byinhibiting one or more of the steps in the Rev pathway described above.They could also inhibit steps downstream of these in the pathway leadingto virus particle assembly and release. The compounds are thereforelikely to form the chemical basis for new drugs that could be used forthe treatment of AIDS.

Development of Cell Lines

The present invention is directed to a safe quantitative in vitrohigh-throughput assay to screen library compounds for effects onRev-dependent p24 production. Two cell lines derived from COS cellsprovide the means of determining whether a library compound has anti-Revactivity-5BD.1 and 2A.22. These cell lines constitutively expressHIV-like particles that contain the HIV core proteins as well as HIVenvelope protein. The non-infectious virions created by these cells aresecreted into the media, where a simple p24 ELISA can quantitativelydetermine virion production. Production of these virus-like particles istotally dependent on the expression of the Rev protein (for the 5BD.1cell line), which is also made in these cell lines, or independent ofRev protein expression (the 2A.22 cell line).

The cell line 5BD.1 was created by transfecting COS cells with the wildtype HIV-1 structural and regulatory genes gag, gagpol, rev, and env.Each of these genes is necessary but not sufficient for producinginfectious HIV virions. Non-infectious virions are produced in 5BD.1cells via the same pathways as in CD4⁺ cells Inhibition of Rev with alibrary compound would therefore have the same effect on viralproduction in 5BD.1 cells as in CD4⁺ cells.

Previous work led to the identification of a small RNA element, namedthe CTE, from Mason-Pfizer Monkey Virus. When this element is present inthe RNA that is expressed from a gene that normally requires Revco-expression the need for Rev is overcome (U.S. Pat. No. 5,880,276,issued Mar. 9, 1999; U.S. Pat. No. 5,585,263, issued Dec. 17, 1996; Brayet al., (1994) Proc. Natl. Acad. Sci. (USA) 91:1256-1260).

Using the CTE as a component of expression vectors has allowed thecreation of a series of expression vectors, which allows expression ofHIV proteins in a Rev-independent fashion. The vectors were then used tocreate stable cell lines that expressed the proteins. One cell line inparticular has proved extremely useful. It is called 2A.22(Srinivasakumar et al. (1997) J. Virol 71:5841-5848). The cell lineexpressed HIV proteins (Gag-GagPol and Envelope) in a Rev-independentfashion.

The Rev-independence of 2A.22 is useful as a negative control whiletesting library compounds. When both cell lines 5BD.1 and 2A.22 aregrown and tested with the same compound under similar conditions, areduction in viral production in 5BD.1 and not in 2A.22 indicates apotentially positive score for that compound as a Rev-specificinhibitor. Alternately, if a compound reduced p24 levels in both 5BD.1and 2A.22, this could indicate a possible harmful interaction withcellular machinery and would rule out that specific compound fromfurther studies.

The non-infectious HIV-like particles created by these cells aresecreted into the media, where a simple p24 ELISA can quantitativelydetermine particle production.

Methods— Anti-Rev Screening to Identify Compounds

The following steps were performed in a drug screening assay of that ledto the identification of the nine parental compounds of the inventiondescribed herein.

Approximately 40,000 compounds (commercially available from SPECS andBioSPECS, Rijswijk, The Netherlands) were screened in this fashion.

Tissue Culture Assay:

-   1. 5BD.1 cells were passaged in 2 T225 flasks in medium (IMDM/10%    FCS/0.2 mg/ml HygromycinB/1.5 mg/ml G418/0.05 mg/ml gentamycin).    Cells were harvested from 90% confluent flasks with 28.4×10⁶ cells    recovered.-   2. 4500 cells per well were plated into columns of tissue culture    treated clear 384 well plates in 40 μl per well of medium.-   3. The plate was placed into the incubator for one hour.-   4. Compounds were diluted from 2 μl of 1 mM DMSO stocks in 384 well    polypropylene plates by adding 38 μl per well of medium.-   5. 10 μl of each diluted compound was transferred to the cell    plates.-   6. The plates were then incubated overnight for 16 hours.-   7. In the morning of the next day the plates were aspirated on a    plate washer.-   8. 40 μl of fresh medium was added to each well followed by 10 μl of    diluted as in step 5.-   9. The plates were then incubated for 8 hours and the supernatant    was collected for measurement in the p24 ELISA Assay.

p24 ELISA Assay:

-   10. Dilute primary antibody to 4 μg/ml in DPBS without calcium and    magnesium, add 25 μl per well of a 384 well Maxisorp plate, incubate    overnight at 4° C.-   11. Aspirate coating solution, block for 30-60 minutes with 100 μl    ELISA buffer (4 mg/ml BSA, 0.01% Tween20 in DPBS without calcium and    magnesium).-   12. Wash plates 2×.-   13. Add 25 μl of supernatant from step 9 above.-   14. Add 10 μl of a 1:250 dilution of biotinylated secondary antibody    in 25% lysis buffer/ELISA buffer.-   15. Incubate overnight in the refrigerator.-   16. Wash plates 3×.-   17. The plates were read in a plate reader at 450 nm.

Example 1

Compounds were identified using the primary screening assay thatinvolved the use of a cell line (5BD.1) that was continuously expressingHIV virus-like particles. To measure inhibition, supernatants containingHIV virus-like particles were obtained from the tissue culture assaydescribed above. The amount of HIV particles in the assay was thenmeasured using the ELISA Assay that is also described above.Representative data showing the final ELISA data from the screening of4,000 of approximately 40,000 compounds screened (commercially availablefrom SPECS and BioSPECS, Rijswijk, The Netherlands) are shown in FIG. 2.192 compounds were identified in this fashion.

Dose Response Assay in 5BD.1 Cells,

Each of the 192 compounds was then subjected to 3 and 6 point doseresponse assays in 5BD.1 cells and toxicity testing in the MT-4 T cellline. The dose response assay was performed as follows

3 and 6 Point Dose Response Assays: Tissue Culture

-   1. 5BD.1 cells were carried in 2 T175 flasks in medium (IMDM/10%    FCS/0.2 mg/ml HygromycinB/1.5 mg/ml G418/0.05 mg/ml gentamycin).    Cells were trypsinized and harvested from a 90% confluent flask.-   2. 20,000 cells per well were plated into rows A-G of 8 tissue    culture treated clear 96-well plates in 135 μl per well of medium    without G418 (assay medium). 135 μl of medium only was added to row    H.-   3. The plates were placed into the incubator for one hour.-   4. Compounds were serially diluted 1:3 two times from 1 mM DMSO    stocks in DMSO in 96 well polypropylene plates. 8 μl of the DMSO    solutions were transferred to another plate and 72 μl of assay    medium was added.-   5. 15 μl of each diluted compound was transferred to the cell plates    in duplicate with the high (10 μM final concentration in rows A-B, 3    μM concentration in rows C-D, 1 μM concentration in rows E-F and    DMSO only in rows G-H).-   6. The plates were then incubated overnight for 16 hours.-   7. In the morning of the next day the medium was removed by hand    from all wells.-   8. 135 μl of fresh assay medium was added to each well followed by    15 μl of diluted compounds as described in step 5.-   9. The plates were then incubated for 24 hours and the medium was    harvested.

p24 ELISA Assay:

-   10. Dilute primary antibody to 4 μg/ml in DPBS without calcium and    magnesium, add 50 μl per well of a 96 well Maxisorp plate, incubate    overnight at 4° C.-   11. Aspirate coating solution, block for 30-60 minutes with 200 μl    ELISA buffer (4 mg/ml BSA, 0.01% Tween20 in DPBS without calcium and    magnesium).-   12. Wash plates 2×.-   13. Add 50 μl of the supernatants from step 9 above.-   14. Add 15 μl of a 1:750 dilution of biotinylated secondary antibody    in 40% lysis buffer/ELISA buffer.-   15. Incubate 2 hours at room temperature with shaking.-   16. Wash plates 3×.-   17. Add 50 μL/well of a 1:10,000 dilution of detection SA-HRP.    Incubate at room temperature with shaking for 30 minutes.-   18. Wash plates 3×. Add 50 μL/well of TMB substrate solution to all    wells and develop for approximately 15 minutes until blue.-   19. Stop the reaction with 50 μL/well 0.18M sulfuric acid.-   20. Read plate at 450 nm.

Toxicity Assays:

MTS-based toxicity assays were performed in parallel to the 3 and 6point dose response assays. The assay uses MTS a tetrazolium compound[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS(b)] and an electron coupling reagent (phenazineethosulfate; PES) and was performed according to the direction of itsmanufacturer Promega, Madison Wis. (see protocol Technical Bulletin #245from Promega).

Example 2

The six point dose assay in 5BD.1 cells was used to calculate an IC₅₀for the compounds and data from the MT-4 toxicity assay allowed a TC₅₀to be calculated. Data for the eight compounds being claimed in thisapplication is given in Table 1. Representative dose response curvesthat were used to calculate these data are illustrated for two of thecompounds in FIG. 3. The name and chemical structure of the ninecompounds are provided in FIG. 4.

TABLE 1 Six point dose response and toxicity assay in 5BD.1 cells andMT4 cells Compound IC₅₀ TC₅₀ TI Code (μM) (μM) (TC₅₀/IC₅₀) 751683.0 >100 >33.3 89246 0.9 20 22.22 91161 1.9 18 9.47 103833 0.9 32 35.6104366 2.5 28 11.2 107129 0.25 10 40.0 107740 4.9 50 10.2 109020 3.0 10033.3

Rev Assay

The eight compounds listed in Table 1 were also tested in an assay thatmeasured HIV-1 Rev function. The HIV-1 Rev assay is based upon the useof a reporter for detecting whether drug candidates are capable ofinhibiting the function of the viral Rev protein. The reporter used forthese assays is the Renilla Luciferase protein, which is an enzyme thatproduces detectable light when mixed with certain chemicals. For thisassay, a cell line has been developed in which the production of thereporter by the cells requires the function of HIV-1 Rev. Therefore, ifa drug candidate inhibits the function of Rev, it decreases the amountof the reporter produced by the cells. By using Renilla Luciferase asthe reporter, the inhibition of Rev is easily detected as a decrease inthe amount of light produced by the cells when mixed with theappropriate chemicals. Furthermore, the cell line for this assay hasbeen engineered to use a second similar reporter (Firefly Luciferase)that detects whether drug candidates are toxic. By using this tworeporter, or Dual-Luciferase, approach, compounds that specificallyinhibit HIV-1 Rev can be identified. More detailed information about theassay is provided in the table 2 below.

TABLE 2 Details of the HIV-1 Rev Assay Parameter HIV-1 Rev Assay AssayPrincipal HIV-1 Rev-dependent Luciferase reporter expression constructengineered into a stable cell line. Reference for Assay Hope et al.(1990) Proc. Natl. Acad. Sci. USA 87: 7787-7791. Principal Cell LineHeLa (cervix; epithelial; adenocarcinoma) Genetic Stably integratedbicistronic expression construct for both the HIV-1_(IIIB) RevModifications gene and Firefly Luciferase gene under the control of asingle Tet-Off promoter; Stably integrated HIV-1_(SF2) Rev-dependentRenilla Luciferase reporter expression construct for monitoring Revfunction. Cell Line DMEM supplemented with 10% Tet-Free FBS,L-Glutamine, Pen/Strep, Maintenance Media Geneticin (G418), Hygromycin Band Puromycin. Passage Trypsinized and split 1:5 twice weekly; Freshcells thawed from LN₂ storage routinely to minimize loss of reportergene expression upon serial passage. Assay Media DMEM supplemented with10% Tet-Free FBS, L-Glutamine, Pen/Strep. Standard Assay 2 × 10⁴cells/well; 96 well format. Conditions Drugs tested at 6 concentrationsin triplicate assay wells. Cells and drug added to wells in 200 μL totalvolume. Plates incubated for 24 hours at 37° C. in humidified 5% CO₂atmosphere. Drugs/media removed, cells lysed and assayed forDual-Luciferase ® reporter expression according to manufacturers kitinstructions (Promega, Madison, WI). Endpoint Detection FireflyLuciferase: luminescence (relative light units) for detection of Tatexpression and compound cytotoxicity/non-specificity Renilla Luciferase:luminescence (relative light units) for detection of compound inhibitionof Rev function Assay Controls Doxycycline: turns off Tet-Off promoterto shut down both Firefly and Renilla Luciferase Expression and verifyassay system functioning properly Leptomycin B: Positive control;inhibitor of hCRM1 mediated Rev nuclear export. Other positive controls:Currently being identified and tested. Data Analysis Calculations ofcompound efficacy for inhibiting Rev function as 50% inhibition ofRenilla Luciferase (IC₅₀), compound toxicity/non-specificity as 50%inhibition of Firefly Luciferase (TC₅₀) and Therapeutic Index (TI =TC₅₀/IC₅₀).

Example 3

Compounds were tested in the Rev assay using the maximum high-testconcentration possible based on the supplied stocks as shown in Table 3.

Compounds were prepared at a 2× high-test concentration by combiningdrug stock with tissue culture media at a ratio of 5 μL of drug to 995μL of media. This 2× high-test sample was subsequently serially dilutedin tissue culture media using ½ log dilutions. This series of 2×concentrated drug was subsequently diluted 1:1 by combining with anequal volume of cells in media in the 96-well plates used for the assay.The results of the assay for all eight compounds are shown in Table 4.Representative data for two of the compounds is also depicted in FIG. 5.

TABLE 3 Compound Information Stock High-Test Concentration ConcentrationCompound Provided (mM) Used (μM) 75168 10.7 26.75 89246 21.8 54.5 9116115.8 39.5 103833 15.04 37.6 104366 31.8 79.5 107129 21.2 53.0 10774011.7 29.25 109020 16.3 40.75

TABLE 4 Rev Assay Results for Compounds Compound IC₅₀ TC₅₀ TI Code (μM)(μM) (TC₅₀/IC₅₀) 75168 2.42 7.91 3.27 89246 0.48 8.41 17.52 911619.73 >39.5 >4.06 103833 1.07 >37.6 >35.14 104366 2.24 17.2 7.68 1071297.48 30.9 4.13 107740 8.00 18.5 2.31 109020 8.98 >40.8 >4.54 LeptomycinB 0.00822 0.0200 2.43 Leptomycin B 0.00817 0.0192 2.35

It should be noted that “Toxicity” in this assay reflects non-specificcompound activity in the assay as demonstrated by a reduction in theFirefly Luciferase signal, which is Rev-independent. This does notnecessarily correlate with cell killing. Leptomycin B is a controlcompound known to inhibit Rev function.

Anti-HIV Efficacy Evaluation in Fresh Human Peripheral Blood MononuclearCells (PBMCs)

Compounds were evaluated for their ability to inhibit HIV replication infresh human PBMCs. The assay was performed as follows.

Preparation of PBMCs

Fresh human PBMCs, seronegative for HIV and HBV, were isolated fromscreened donors (Interstate Blood Bank, Inc. Memphis, Tenn.). Cells werepelleted/washed 2-3 times by low speed centrifugation and re-suspensionin PBS to remove contaminating platelets. The Leukophoresed blood wasthen diluted 1:1 with Dulbecco's Phosphate Buffered Saline (DPBS) andlayered over 14 mL of Lymphocyte Separation Medium (LSM; Cellgro® byMediatech, Inc.; density 1.078+/−0.002 g/ml; Cat.# 85-072-CL) in a 50 mLcentrifuge tube and then centrifuged for 30 minutes at 600×g. BandedPBMCs were gently aspirated from the resulting interface andsubsequently washed 2× with PBS by low speed centrifugation. After thefinal wash, cells were enumerated by trypan blue exclusion andre-suspended at 1×10⁷ cells/mL in RPMI 1640 supplemented with 15% FetalBovine Serum (FBS), and 2 mM L-glutamine, 4 μg/mL Phytohemagglutinin(PHA-P, Sigma). The cells were allowed to incubate for 48-72 hours at37° C. After incubation, PBMCs were centrifuged and resuspended in RPMI1640 with 15% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mLstreptomycin, 10 μg/mL gentamycin, and 20 U/mL recombinant human IL-2(R&D Systems, Inc). IL-2 is included in the culture medium to maintainthe cell division initiated by the PHA mitogenic stimulation. PBMCs weremaintained in this medium at a concentration of 1−2×10⁶ cells/mL withbiweekly medium changes until used in the assay protocol. Cells werekept in culture for a maximum of two weeks before being deemed too oldfor use in assays and discarded. Monocytes were depleted from theculture as the result of adherence to the tissue culture flask.

Viral Assay

For the standard PBMC assay, PHA-P stimulated cells from at least twonormal donors were pooled (mixed together), diluted in fresh medium to afinal concentration of 1×10⁶ cells/mL, and plated in the interior wellsof a 96 well round bottom microplate at 50 (5×10⁴ cells/well) in astandard format developed by the Infectious Disease Research departmentof Southern Research Institute. Pooling (mixing) of mononuclear cellsfrom more than one donor is used to minimize the variability observedbetween individual donors, which results from quantitative andqualitative differences in HIV infection and overall response to the PHAand IL-2 of primary lymphocyte populations. Each plate containsvirus/cell control wells (cells plus virus), experimental wells (drugplus cells plus virus) and compound control wells (drug plus mediawithout cells, necessary for MTS monitoring of cytotoxicity). SinceHIV-1 is not cytopathic to PBMCs, this allows the use of the same assayplate for both antiviral activity and cytotoxicity measurements. Testdrug dilutions were prepared at a 2× concentration in microtiter tubesand 100 μL of each concentration was placed in appropriate wells usingthe standard format. 50 μL of a predetermined dilution of virus stockwas placed in each test well (final MOI≅0.1). The PBMC cultures weremaintained for seven days following infection at 37° C., 5% CO₂. Afterthis period, cell-free supernatant samples were collected for analysisof reverse transcriptase activity as a measure of virus release.Following removal of supernatant samples, compound cytotoxicity wasmeasured by addition of MTS to the plates for determination of cellviability. Wells were also examined microscopically and anyabnormalities were noted.

Reverse Transcriptase Activity Assay

A microtiter plate-based reverse transcriptase (RT) reaction wasutilized (Buckheit et al., AIDS Research and Human Retroviruses7:295-302, 1991). Tritiated thymidine triphosphate (³H-TTP, 80 Ci/mmol,NEN) was received in 1:1 dH₂O:Ethanol at 1 mCi/mL. Poly rA:oligo dTtemplate:primer (Pharmacia) was prepared as a stock solution bycombining 150 μL poly rA (20 mg/mL) with 0.5 mL oligo dT (20 units/mL)and 5.35 mL sterile dH₂O followed by aliquoting (1.0 mL) and storage at−20° C. The RT reaction buffer was prepared fresh on a daily basis andconsisted of 125 μL 1.0 M EGTA, 125 μL dH₂O, 125 μL 20% Triton X100, 50μL 1.0 M Tris (pH 7.4), 50 μL 1.0 M DTT, and 40 μL 1.0 M MgCl₂. Thefinal reaction mixture was prepared by combining 1 part ³H-TTP, 4 partsdH₂O, 2.5 parts poly rA:oligo dT stock and 2.5 parts reaction buffer.Ten microliters of this reaction mixture was placed in a round bottommicrotiter plate and 15 μL of virus containing supernatant was added andmixed. The plate was incubated at 37° C. for 60 minutes. Followingincubation, the reaction volume was spotted onto DE81 filter-mats(Wallac), washed 5 times for 5 minutes each in a 5% sodium phosphatebuffer or 2×SSC (Life Technologies). Next, they were washed 2 times for1 minute each in distilled water, 2 times for 1 minute each in 70%ethanol, and then dried. Incorporated radioactivity (counts per minute,CPM) was quantified using standard liquid scintillation techniques.

MTS Staining for PBMC Viability to Measure Cytotoxicity

At assay termination, assay plates were stained with the solubletetrazolium-based dye MTS (CellTiter 96 Reagent, Promega) to determinecell viability and quantify compound toxicity. The mitochondrial enzymesof metabolically active cells metabolize MTS to yield a soluble formazanproduct. This allows the rapid quantitative analysis of cell viabilityand compound cytotoxicity. The MTS is a stable solution that does notrequire preparation before use. At termination of the assay, 20 μL ofMTS reagent was added per well. The microtiter plates were thenincubated 4-6 hrs at 37° C. The incubation intervals were chosen basedon empirically determined times for optimal dye reduction. Adhesiveplate sealers were used in place of the lids, the sealed plate wasinverted several times to mix the soluble formazan product and the platewas read spectrophotometrically at 490/650 nm with a Molecular DevicesVmax or SpectraMaxPlus plate reader.

Virus Information

The HIV-1 clinical isolate BR/93/021 was obtained from the NIH AIDSResearch and Reference Reagent Program. This virus was originallyisolated from a seropositive individual in Brazil and has beencharacterized as an Envelope Subtype B and as an R5-tropic isolate. TheHIV-1 molecular clone NL4-3 was obtained from the NIH AIDS Research andReference Reagent Program. Virus stocks were prepared by transfection ofplasmid DNA into cells using standard techniques. A Pre-titered aliquotof each virus was removed from the freezer (LN₂) and thawed rapidly toroom temperature in a biological safety cabinet immediately before use.

Example 4

Compounds were tested in the PBMC assay using the maximum high-testconcentration possible based on the supplied stocks which are listed intable 3. Compounds were prepared at a 2× high-test concentration bycombining drug stock with tissue culture media at a ratio of 5 μL ofdrug to 995 μL of media. This 2× high-test sample was subsequentlyserially diluted in tissue culture media using 1/2 log dilutions. Thisseries of 2× concentrated drug was subsequently diluted 1:1 by combiningwith an equal volume of cells in media in the 96-well plates used forthe assay. The results of the assay for all eight compounds are shown inTable 5. Representative data for two of the compounds with each virusare shown in FIGS. 6 and 7.

TABLE 5 Results for Compounds Against HIV-1 in Fresh Human PBMCs HIV-1BR/93/021 HIV-1 NL4-3 TI TI IC₅₀ TC₅₀ (TC₅₀/ IC₅₀ TC₅₀ (TC₅₀/ Compound(μM) (μM) IC₅₀) (μM) (μM) IC₅₀) 75168 2.14 >26.8 >12.52 16.9 >26.8 >1.5989246 0.47 32.7 69.57 1.47 21.9 14.90 91161 1.94 23.3 12.01 2.28 13.55.92 103833 0.29 >37.6 >129.66 2.22 >37.6 >16.94 104366 0.64 24.9 38.910.92 22.0 23.91 107129 1.80 6.26 3.48 2.12 4.13 1.95 107740 0.20 18.894.00 1.61 14.4 8.94 109020 13.2 >40.8 >3.09 26.4 >40.8 >1.55 AZT0.00247 >1.0 >404.86 0.00062 >1.0 >1612.90 AZT was used as an anti-viralcontrol.Efficacy Evaluation in U1 Cells Latently Infected with HIV-1 U1 CellCulture

U1 cells were obtained from the AIDS Research and Reference ReagentProgram and maintained under standard culture conditions in RPMI 1640supplemented with 10% fetal bovine serum (heat inactivated), 2 mML-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. U1 cellsare derived from the histocytic leukemia cell line U937, and contain anintegrated copy of a cytokine and/or phorbol inducible provirus(HIV-1_(IIIB)). Cultures were maintained in such a way as to ensureexponential growth of the populations. Addition of TNF-α to the cellculture induces HIV-1, so that inhibition of virus production indicatesan inhibition of a post-integration function of the HIV-1 life cycle.This is where an inhibitor of Rev function would be expected to act.Cells were collected by centrifugation and counted by hemacytometer

Assay Setup

At the time of the assay, cells were collected by centrifugation andcounted by hemacytometer. If cell viability by Trypan Blue dye exclusionwas less than 70% the assay was terminated. The cells were adjusted to5×10⁴ cells/mL and 100 μL placed in 96 well plates with 100 μL mediacontaining a final concentration of 5 ng/mL TNF-α and the test compound.Cultures were incubated for 3 days and supernatants harvested. Compoundtoxicity was determined by MTS dye reduction. Virus expression wasmeasured by supernatant reverse transcriptase activity.

Reverse Transcriptase Activity Assay

Reverse transcriptase was assayed to measure virus particle release asdescribed above for the PBMC assay.

MTS Staining for Cell Viability

At assay termination, the assay plates were stained with the solubletetrazolium-based dye MTS (CellTiter 96 Reagent, Promega) to determinecell viability and quantify compound toxicity as described for the PBMCassay.

Example 5

Compounds were tested in the U1 latency assay using the maximumhigh-test concentration possible based on the supplied stocks which arelisted in table 3. Compounds were tested using the maximum high-testconcentration possible based on the supplied stocks. Compounds wereprepared at a 2× high-test concentration by combining drug stock withtissue culture media at a ratio of 54 of drug to 995 μL of media. This2× high-test sample was subsequently serially diluted in tissue culturemedia using ½ log dilutions. This series of 2× concentrated drug wassubsequently diluted 1:1 by combining with an equal volume of cells inmedia in the 96-well plates used for the assay. Temacrazine a knowninhibitor of HIV transcription was used as a control compound that isactive in the post-integration part of the HIV life cycle.

The results of the assay for seven of the eight compounds are shown inTable 6. Representative data for two of the compounds and the controlare shown in FIG. 8.

TABLE 6 Results for Compounds in U1 Cell Assay IC₅₀ TC₅₀ TI Compound(μM) (μM) (TC₅₀/IC₅₀) 89246 0.24 24.6 102 91161 16.5 22.3 1.35 1038330.40 >37.6 >94 104366 <0.25 12.4 >49.6 107129 >53.0 >53.0 N/A 1077401.1 >29.3 >26.6 109020 0.27 >40.8 >151 Temacrazine 0.002 >0.5 >250

Analogs of the Parental Compounds

Analog searches of 7 of the parental compounds were performed on theSpecs database of compounds using ISIS software from MDL InformationSystems Inc. (Elsevier MDL, 14600 Catalina Street San Leandro, Calif.94577). Analogs of each of the seven compounds were found and are shownin FIGS. 9-15. The following number of analogs were found for eachcompound.

TABLE 7 Analogs for each compound Compound Number of analogs 89246 491161 13 103833 29 104366 6 107129 7 107740 6 109020 18Compounds were tested using the maximum high-test concentration possiblebased on the supplied stocks. Compounds were prepared at a 2× high-testconcentration by combining drug stock with tissue culture media at aratio of 5 μL of drug to 995 μL of media. This 2× high-test (HT) samplewas subsequently serially diluted in tissue culture media using ½ logdilutions. This series of 2× concentrated drug was subsequently diluted1:1 by combining with an equal volume of cells in media in the 96-wellplates used for the assay. The results are shown in Table 8.

TABLE 8 Results for Analogs in U1 Cell Assay Compound HT Conc. IC₅₀ TC₅₀TI 89246  54.5 μM <0.172 μM 29.0 μM >169 111 50.0 μM 1.70 μM >50.0μM >29.4 112 50.0 μM 1.08 μM >50.0 μM >46.3 113 75.0 μM 27.4 μM >75.0μM >2.74 114 75.0 μM 14.4 μM >75.0 μM >5.21 104366 211 75 μM 9.11 μM48.5 μM 5.32 212 50 μM 0.484 μM 28.6 μM 59.1 213 75 μM 0.826 μM 58.6 μM70.9 214 75 μM 9.19 μM >75.0 μM >8.16 215 37.5 μM 32.2 μM >37.5 μM >1.16216 75 μM 2.73 μM 13.0 μM 4.76 103833 311 75 μM 1.65 μM >75.0 μM >45.5312 75 μM 7.13 μM >75.0 μM >10.5 313 50 μM >50.0 μM >50.0 μM N/A 314 75μM 2.11 μM >75.0 μM >35.5 315 75 μM 0.750 μM >75.0 μM >100 316 75 μM23.4 μM >75.0 μM >3.21 317 75 μM >75.0 μM >75.0 μM N/A 318 75 μM <0.237μM >75.0 μM >316 319 37.5 μM 20.2 μM >37.5 μM >1.86 320 75 μM 0.522μM >75.0 μM >144 321 75 μM 1.94 μM >75.0 μM >38.7 322 75 μM 8.84 μM 17.4μM 1.97 323 75 μM 0.745 μM 22.6 μM 30.3 324 75 μM 2.19 μM >75.0 μM >34.2325 75 μM 0.281 μM >75.0 μM >267 326 75 μM <0.237 μM 52.8 μM >223 327 75μM 0.601 μM 65.8 μM 109 328 75 μM 1.06 μM 37.0 μM 34.9 329 75 μM 2.03 μM58.4 μM 28.8 330 75 μM 6.91 μM >75.0 μM >10.9 331 75 μM 66.0 μM >75.0μM >1.14 332 75 μM >75.0 μM 22.5 μM <0.30 333 20 μM >20.0 μM >20.0 μMN/A 334 75 μM 2.37 μM >75.0 μM >31.6 335 75 μM 1.41 μM 53.9 μM 38.2 33675 μM 2.92 μM >75.0 μM >25.7 337 75 μM 2.40 μM >75.0 μM >31.3 338 75 μM5.23 μM >75.0 μM >14.3 339 75 μM 1.28 μM 38.3 μM 29.9 91161 411 75μM >75.0 μM >75.0 μM N/A 412 75 μM 16.7 μM 51.7 μM 3.10 413 75 μM 20.7μM 50.7 μM 2.45 414 75 μM 14.5 μM 14.7 μM 1.01 415 75 μM 37.3 μM 51.2 μM1.37 416 75 μM 0.767 μM >75.0 μM >97.8 417 75 μM 1.62 μM 51.9 μM 32.0418 75 μM 2.18 μM >75.0 μM >34.4 419 75 μM 1.86 μM 49.4 μM 26.6 420 75μM 6.80 μM >75.0 μM >11.0 421 75 μM >75.0 μM >75.0 μM N/A 422 75 μM 14.3μM >75.0 μM >5.24 423 75 μM 42.4 μM >75.0 μM >1.77 107740 511 75 μM 14.3μM 17.3 μM 1.21 512 75 μM 26.3 μM >75.0 μM >2.85 513 75 μM 7.05 μM >75.0μM >10.6 514 75 μM >75.0 μM >75.0 μM N/A 515 75 μM 13.6 μM >75.0μM >5.51 516 6.25 μM >6.25 μM >6.25 μM N/A 107129 611 75 μM 17.5 μM 15.0μM 0.86 612 75 μM >75.0 μM >75.0 μM N/A 613 75 μM >75.0 μM >75.0 μM N/A614 75 μM >75.0 μM >75.0 μM N/A 615 75 μM 11.9 μM >75.0 μM >6.30 616 75μM 0.915 μM >75.0 μM >82.0 617 75 μM >75.0 μM >75.0 μM N/A 618 75 μM18.3 μM 53.6 μM 2.93 109020 718 75 μM 47.2 μM 50.1 μM 1.06 719 75 μM1.73 μM >75.0 μM >43.4 720 75 μM 3.66 μM >75.0 μM >20.5 722 75 μM 21.9μM 35.0 μM 1.60 728 75 μM 13.4 μM 17.5 μM 1.31 729 75 μM >75.0 μM >75.0μM N/A 730 75 μM >75.0 μM >75.0 μM N/A 731 75 μM 1.65 μM >75.0 μM >45.5734 12.5 μM 4.07 μM >12.5 μM >3.07 748 75 μM 10.5 μM 33.0 μM 3.14 749 75μM 2.05 μM 19.5 μM 9.51 750 75 μM >75.0 μM >75.0 μM N/A 751 75 μM 71.4μM >75.0 μM >1.05 752 75 μM >75.0 μM >75.0 μM N/A 753 75 μM 47.1 μM 48.0μM 1.02 754 75 μM 3.69 μM 15.1 μM 4.09 755 75 μM 1.78 μM >75.0 μM >42.1756 75 μM 1.74 μM 52.0 μM 29.9 Temacrazine 500 nM 1.56 nM >500 nM >320Temacrazine 500 nM 3.84 nM >500 nM >130

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated by reference herein intheir entirety.

One of skill in the art will appreciate that the superiority of thecompositions and methods of the invention relative to the compositionsand methods of the prior art are unrelated to the physiological accuracyof the theory explaining the superior results.

Headings are included herein for reference and to aid in locatingcertain sections. These headings are not intended to limit the scope ofthe concepts described therein under, and these concepts may haveapplicability in other sections throughout the entire specification.

The previous description of the disclosed embodiments is provided toenable any person skilled in the art to make or use the presentinvention. Various modifications to these embodiments will be readilyapparent to those skilled in the art, and the generic principles definedherein may be applied to other embodiments without departing from thespirit or scope of the invention. The appended claims are intended to beconstrued to include all such embodiments and equivalent variations.Accordingly, the present invention is not intended to be limited to theembodiments shown herein but is to be accorded the widest scopeconsistent with the principles and novel features disclosed herein.

1-24. (canceled)
 25. A method of inhibiting HIV replication, said methodcomprising contacting a cell comprising HIV with an effective amount ofa compound selected from the group consisting of:


26. The method of claim 25, wherein said compound inhibits REV function.27. The method of claim 25, wherein HIV virion production is dependenton Rev protein expression.